Although many of the pleiotropic responses of cells to the phorbol ester tumor promoters have been described and a putative receptor identified, a biochemical mechanism for the regulation of these tumor promoter induced pleiotropic responses has not been elucidated. We propose that phorbol ester induced phospholipid turnover is related to the induction of increased phospholipid hydrolysis. This hydrolysis causes the accumulation of 1, 2 diacylglycerol formation in cellular membranes. The increased 1, 2 diacyl-glycerol level could cause a decrease in Ka for Ca++ ions of a newly described Ca++ phospholipid dependent protein kinase (protein kinase C). The result of this would be the enhanced phosphorylation of critical cellular proteins involved inthe pleiotropic response to these tumor promoters. We have already been able to detect an enhanced turnover of phosphatidylcholine, choline and phosphorycholine release, and 1, 2 diacylglycerol accumulation in C3H/10T1/2 cells within 10 min. after the addition of 12-0-tetradecanoyl phorbol-13-acetate (TPA) to these cells. We intend to further characterize the accumulation of 1, 2 diacylgylcerol in response to other phorbol esters and compounds which mimic the effects of phorbol esters, but which do not bind to the phorbol ester receptor. A determination of which membrane fraction this increased level of 1, 2 diacylglycerol occurs in will also be made. We will also examine C3H/10T1/2 cells for the presence of Ca++ phospholipid dependent protein kinase which is responsive to 1, 2 diacylglycerol. The enzyme will be partially purified and characterized. The response of this protein kinase in vitro to the increased levels of diacylglycerol in TPA treated membranes will also be determined. In addition, we will determine if TPA activates the phosphorylation of any endogenous substrates in intact cells using SDS polyacrylamide gel electrophoresis. We will identify these substrates and determine if they can act as substrates for protein kinase C in vitro. Finally, we will determine if treatment of C3H/10T1/2 with exogenous phospholipase C can mimic the affects of phorbol esters (e.g., accelerated phosphatidylcholine turnover, and induction of the serine protease plasminogen activator). The results of this proposal should lead to a clearer understanding of the biochemical mechanisms that tumor promoters activate to alter the cellular phenotype and ultimately enhance carcinogenesis.